Objective:

Environmentally benign alternatives to chemical insecticides are required
to reduce the deleterious impact of these agents on nontarget organisms.
Insect
viruses, i.e. baculoviruses, are one of the most promising biological agents
since they are relatively host specific and do not adversely affect the
environment. The long-term goal of the proposed collaborative research
is to develop a
more cost-effective method for mass-production of baculovirus insecticides.
This method involves continuous production of baculoviruses in cell culture
without production of few polyhedra (FP) mutant accumulation. FP mutant
accumulation results from mutation of the baculovirus fp25k gene that results
in loss
of FP25K protein expression. Transposon insertion at specific sites in
fp25k results in the FP genotype. The hypothesis to be tested in the Bonning
lab
is that FP mutant accumulation in cell culture can be overcome by modification
of the baculovirus gene fp25k. The goal of this research is to generate
a genetically stable virus to prevent the negative impact of serial passage
on the baculovirus Autographa californica multiple nucleopolyhedrovirus
(AcMNPV).

Approach:

Based on the fact that transposon insertion into
the fp25k gene occurs at TTAA target sites, we plan to remove
the TTAA sites by site-directed mutagenesis. We will test the hypothesis
that disruption
of fp25k by transposon insertion will be prevented by mutation of the TTAA
target sites. Following production of the modified baculovirus and preliminary
characterization
in the Bonning lab, the viruses will be amplified and tested for stability
on repeated passaging in vitro and in the continuous bioreactor system in
theMurhammer
lab.

Expected Results:

We expect that removal of TTAA target sites from AcMNPV
fp25k will stabilize the virus on repeated passaging in cell culture, by
preventing insertion of transposable elements from the host cells into the
fp25k gene.
All of the modifications that cause FP production in AcMNPV can be explained
by transposon activity. Acquisition of a virus that is stable in a continuous
bioreactor system will be a key step toward use of this cost-effective method
for large-scale production of baculovirus insecticides. Increased use of
baculovirus insecticides in integrated pest management will help minimize economic
losses
resulting from insect pests, without the negative environmental impact associated
with classical chemical insecticides.

Successful completion of this project will pave the way for cost-effective
large scale in vitro production of baculovirus insecticides. This method will
increase the likelihood that baculovirus insecticides will be marketed and
used more widely for management of agricultural insect pests, thereby reducing
the amount of chemical insecticides required for pest management.

Progress and Final Reports:

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.